Genetic information is usually represented as linear arrays on chromosomes, yet nuclear DNA exists in a highly compacted form in which access to individual genes must be considered as a three-dimensional problem. Cytological evidence for a limited number of loci in several eukaryotes has suggested that localization of at least some genes is related to the transcriptional activity of the genes, and that this positioning can change in response to metabolic and developmental signals. Using the budding yeast, Saccharomyces cerevisiae, we have recently shown that the 275 tRNA genes, although widely scattered in the linear genome, are gathered near the nucleolus throughout the cell cycle. This clustering of the tRNA genes, while potentially advantageous in terms of RNA polymerase III (pol III) transcriptional regulation and an ordered pre-Trna processing pathway, has striking implications for spatial organization of much of the yeast genome. The clustering is pol III transcription-dependent, and affects both recombinations between tRNA genes and nearby transcription by RNA polymerase II. In preliminary studies we have now shown that nucleolar tRNA gene localization is a two step process, with condensin-dependent clustering of the tRNA genes separable from microtubule-dependent localization of the clusters to the nucleolus. The near-term goal of this project is to characterize the mechanisms of this large-scale organization of the individual tRNA genes in yeast, to take advantage of the molecular genetic tools. In the longer term we will also investigate whether this phenomenon affects the behavior of RNA polymerase III transcription units in mammals. Genomes from humans and other vertebrates contain not only hundreds of tRNA genes, but hundreds of thousands of short interspersed DNA elements (SINEs) with tRNA-class promoters. Some of these short interspersed DNA elements (SINEs) are known to influence both nearby transcription by RNA polymerase II and recombination events that are associated with developmental abnormalities and cancers. We anticipate that understanding the spatial behavior of pol III transcription units could provide broad insights into constraints on genome organization and regulation. PUBLIC HEALTH RELEVANCE: DNA in living cells is extremely condensed and packaged in a highly organized fashion. This packaging facilitates regulated retrieval of genetic information and affects DNA recombination events that can lead to cancers and developmental abnormalities. We have identified a type of abundant DNA element, distributed throughout genomes of higher cells, that both encodes small RNAs and serves as a three-dimensional organization signal. We are investigating the molecular mechanisms of this organization and the nature of the resulting DNA packaging.